The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Recombinant therapeutic proteins are commonly produced in several mammalian host cell lines including murine myeloma NS0 and Chinese Hamster Ovary (CHO) cells (Anderson, D. C and Krummen, L. (2002) Curr. Opin. Biotech. 13: 117-123; Chu, L. and Robinson, D. K. (2001) Curr. Opin. Biotechnol. 12:180-187). Each cell line has advantages and disadvantages in terms of productivity and the characteristics of the proteins produced by the cells. Choices of commercial production cell lines often balance the need for high productivity with the ability to deliver the product quality attributes required of a given product. One important class of therapeutic recombinant proteins which often require high titer processes are monoclonal antibodies. Some monoclonal antibodies need effector functions, mediated through the Fc region, to elicit their biological functions. An example is rituximab (RITUXAN®, Genentech, Inc. and Biogen-Idec), a chimeric monoclonal antibody which binds to cell surface CD-20 and results in B-cell depletion (Cartron et al (2002) Blood 99: 754-758; Idusogie et al (2000) J. Immunol. 164: 4178-4184). Other antibodies, such as bevacizumab (AVASTIN™, Genentech, Inc.), a humanized anti-VEGF (vascular endothelial growth factor) antibody, do not require Fc effector functions for their activity.
Advances in fermentation and cell culture techniques have greatly increased the titers of target proteins in culture fluid. This increase in upstream efficiency has led to a bottleneck in downstream processing at the cell-harvest stage. Cell harvesting, or clarification of the harvested cell culture fluid, is an important process in nearly all downstream purifications of biotech-based products. When the product is internal to the cells, cell harvesting is used to decrease the liquid volume of cells to be processed in the product extraction steps. When the product is extracellular, cell harvesting is used to separate the product from the cells and cellular debris, for example, the isolation of an extracellular antibody from mammalian cell culture (Anthony S. Lubiniecki, Ed. (1990) Large-Scale Mammalian Cell Culture Technology, Marcel Dekker; Hansjoerg Hauser, Roland Wagner, Eds. (1997) Mammalian Cell Biotechnology in Protein Production, Walter Gruyter Publishing).
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.
During the purification of therapeutic antibodies, impurities including host cell proteins, product variants, host cell DNA, small molecules, process related contaminants, endotoxins and viral particles must be removed (Fahrner, R. L. et al (2001) Biotechnol. Genet. Eng. Rev. 18:301-327). The purification techniques used must be scaleable, efficient, cost-effective, reliable, and meet the rigorous purity requirements of the final product. Current purification techniques typically involve multiple chromatographic separations employing orthogonal modes of separation. A typical process might include some of the following steps: precipitation (U.S. Pat. No. 7,169,908), dialysis, electrophoresis, ultrafiltration, affinity chromatography, cation exchange chromatography, anion exchange chromatography and/or hydrophobic interaction chromatography. Conventional column chromatography steps are effective and reliable, but generally have low product throughput (kg processed/h). As monoclonal antibodies become more widely used, more efficient process-scale production is necessary.
Chromatography techniques exploit the chemical and physical properties of proteins to achieve a high degree of purification. These chemical and physical properties typically include size, isoelectric point, charge distribution, hydrophobic sites and affinity for ligands (Janson, J. C. and L. Ryden (eds.). (1989) Protein Purification: Principles, High Resolution Methods and Applications. VCH Publishers, Inc., New York). The various separation modes of chromatography include: ion-exchange, chromatofocusing, gel filtration (size exclusion), hydrophobic interaction, reverse phase, and affinity chromatography. Ion-exchange chromatography (IEX), including anion-exchange and cation-exchange chromatography separates analytes (e.g. proteins) by differences of their net surface charges. IEX is a primary tool for the separation of expressed proteins from cellular debris and other impurities. Today, IEX is one of the most frequently used techniques for purification of proteins, peptides, nucleic acids and other charged biomolecules, offering high resolution and group separations with high loading capacity. The technique is capable of separating molecular species that have only minor differences in their charge properties, for example two proteins differing by one charged amino acid. These features make IEX well suited for capture, intermediate purification or polishing steps in a purification protocol and the technique is used from microscale purification and analysis through to purification of kilograms of product.
Chromatography techniques are reliable but capacity and throughput can be problematic for large scale applications. Conventional column chromatography steps are effective and reliable, but generally have low product throughput (kg processed/h). As recombinant proteins become more widely used, more efficient process-scale production is necessary. The throughput of a chromatography step is typically limited by the capacity of the chromatography resin for the protein of interest. With increased protein load to the column, resolution of the protein of interest from impurities often decreases.
Polyelectrolytes are known to form complexes with proteins which take the forms of soluble complexes (Dellacherie, E. (1991) Am. Chem. Soc., Div. Polym. Chem. Prepr. 32(1):602), amorphous precipitates (Mattiasson et al (1998) Polym. Plast. Technol. Eng. 37(3):303-308; Clark et al (1987) Biotech. Progress 3(4):241; Fisher et al (1988) Biotechnol. Bioeng. 32:777; Shieh et al (1991) Am. Chem. Soc., Div. Polym. Chem. Prepr. 32(1)606; Sternberg et al (1974) Biochimica et Biophysica Acta 342:195-206; WO 2004/014942), or coacervates (Wang et al (1996) Biotechnol. Prog. 12:356-362; Veis, A. (1991) Am. Chem. Soc. Div. Polym. Chem. Prepr. 32(1) 596). Papain proteolysis of monoclonal antibodies in the presence of antigen-polycation (polymethacrylic acid) gives Fab fragments (Dainiak et al (2000) Analytical Biochem. 277:58-66).
Protein-polyelectrolyte complexes coacervate, i.e. separate into two distinct liquid phases where the coacervate phase contains most of the complex and the other phase is the equilibrium phase (Burgess, D. J. “Complex Coacervation: Microcapsule Formation” in Macromolecular Complexes in Chemistry and Biology, Dubin, P. L., et al Eds. (1994) Springer-Verlag, Berlin; Dubin et al (1994) Sep. Purif. Methods 23:1-16). Polyelectrolyte coacervation of proteins is a complicated process and is not useful for a broad range of proteins. The intermolecular associations in protein-polyelectrolyte complexes are due to electrostatic interactions, hydrogen bonds and hydrophobic forces (Cooper et al (2005) Current Opinion in Colloid & Interface Science 10:52-78; Mattison et al (1999) Macromol. Symp. 140:53-76). While it is known that addition of a polyelectrolyte to a protein solution can lead to the formation of protein-polyelectrolyte complexes and larger clusters and eventually to a coacervate and/or precipitation, the reverse process may appear upon further addition of polyelectrolyte whereby redissolution of protein occurs, defeating the attempt at protein isolation or purification (Carlsson et al (2003) J. Am. Chem. Soc. 125:3140-3149). Precipitation of proteins using polyelectrolytes may provide a cost-effective alternative to chromatography separation. Using this technique, it may be possible to exploit the functional chemistry of chromatographic techniques to achieve a similar level of protein purification in solution. In particular, the throughput of the precipitation step would no longer be limited by the capacity of a particular chromatography resin.